GULP1 as a Downstream Effector of the Estrogen Receptor-β Modulates Cisplatin Sensitivity in Bladder Cancer

Abstract

Background/aim: Precise molecular mechanisms underlying resistance to cisplatin-based chemotherapy remain unclear, while the activity of estrogen receptor-β (ERβ) has been suggested to be associated with chemosensitivity in urothelial cancer. We aimed to determine if GULP1, an adapter protein known to facilitate phagocytosis, could represent a downstream effector of ERβ and thereby modulate cisplatin sensitivity in bladder cancer.

Materials and methods: GULP1 expression and cisplatin cytotoxicity were compared in bladder cancer lines. Immunohistochemistry was used to determine the expression of GULP1 and ERβ in two sets of tissue microarray (TMA) consisting of transurethral resection specimens.

Results: The levels of GULP1 expression were considerably higher in ERβ-knockdown sublines than in the respective control ERβ-positive sublines. Estradiol treatment reduced GULP1 expression in ERα-negative/ERβ-positive lines, which was restored by the anti-estrogen tamoxifen. Chromatin immunoprecipitation assay revealed the binding of ERβ to the GULP1 promoter in bladder cancer cells. Moreover, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment, but not to other chemotherapeutic agents, including gemcitabine, methotrexate, vinblastine, and doxorubicin. In the first set of TMA (n=129), the expression of ERβ and GULP1 was inversely correlated (p=0.023), and ERβ(-)/GULP1(+) in 51 muscle-invasive tumors was associated with significantly lower risk of disease progression and cancer-specific mortality. Similarly, in the second set (n=43), patients with ERβ(-)/GULP1(+) muscle-invasive disease were significantly (p=0.021) more likely to be responders to cisplatin-based neoadjuvant chemotherapy before radical cystectomy.

Conclusion: ERβ activation was found to reduce the expression of GULP1 as a direct downstream target in bladder cancer cells, resulting in the induction of cisplatin resistance.

Keywords: Bladder cancer; GULP1; chemotherapy; cisplatin; estrogen receptor-β.

Figure 1

Associations between estrogen receptor-β (ERβ) signaling and GULP1 expression in bladder cancer cells. (A) Western blotting of ERβ and GULP1 in UMUC3-control-shRNA vs. UMUC3-ERβ-shRNA and 5637-control-shRNA vs. 5637-ERβ-shRNA. (B) Western blotting of GULP1 in UMUC3, 5637, UMUC3-ERβ-shRNA, and 5637-ERβ-shRNA cultured for 24 h with ethanol (mock), E2 (10 nM) and/or tamoxifen (TAM; 1 μM), as indicated. GAPDH served as a loading control. (C) The ChIP assay, using UMUC3 cell lysates immunoprecipitated with an anti-ERβ antibody (or IgG as a negative control). DNA fragments were PCR amplified with a set of GULP1 promoter-specific primers, and the PCR products (i.e., 328 bp for the binding site) were electrophoresed on 1% agarose gel. A fraction of the mixture of protein-DNA complex (i.e., 1% of total cross-linked, reserved chromatin prior to immunoprecipitation) was used as “input” DNA.

Figure 2

Effects of GULP1 knockdown on cytotoxicity of cisplatin (CDDP) in bladder cancer cells. MTT assay in UMUC3-control-shRNA and UMUC3-GULP1-shRNA sublines (A), as well as in 647V-control-shRNA and 647V-GULP1-shRNA sublines (B), cultured for 48 h in the absence or presence of 5 μM CDDP. Cell viability is presented relative to that of each subline without drug treatment from triplicate experiments. *p<0.05 (vs. control-shRNA with CDDP).

Figure 3

Effects of GULP1 knockdown on cytotoxicity of other chemotherapeutic drugs in bladder cancer cells. MTT assay in UMUC3-control-shRNA and UMUC3-GULP1-shRNA sublines, as well as in 647V-control-shRNA and 647V-GULP1-shRNA, cultured for 48 h in the absence or presence of 5 μM gemcitabine (GEM) (A), 0.5 μM methotrexate (MTX) (B), 15 μM vinblastine (VBL) (C), or 0.5 μM doxorubicin (DXR) (D). Cell viability is shown relative to that of each subline without drug treatment from triplicate experiments.

Figure 4

Immunohistochemistry of estrogen receptor-β (ERβ) and GULP1 in surgical specimens. (A) Representative images of ERβ and GULP1 expression in bladder cancer (original magnification: 200×). Kaplan-Meier curves for progression-free survival and cancer-specific survival in patients with ERβ-negative/GULP1-positive (n=5) vs. ERβ-positive//GULP1-negative (n=17) muscle-invasive tumors (B) or ERβ-negative/GULP1-positive (n=5) vs. ERβ-positive or ERβ-negative/GULP1-negative (n=46) muscle-invasive tumors (C).